Automation of nanoflow liquid chromatography-tandem mass spectrometry for proteome analysis by using a strong cation exchange trap column
Automation of nanoflow liquid chromatography-tandem mass spectrometry for proteome analysis by using a strong cation exchange trap column
复制标题
使用强阳离子交换捕获柱进行蛋白质组分析的纳流液相色谱-串联质谱自动化
DOI：
10.1002/pmic.200600661
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发表时间：
2007-02-01
期刊：
影响因子：
3.4
通讯作者：
Zou, Hanfa
中科院分区：
文献类型：
--
作者：
Jiang, Xiaogang;Feng, Shun;Zou, Hanfa
建立了一种利用强阳离子交换(SCX)捕捉柱自动进样的纳流LC-MS/MS(MU LC-MS/MS)分析方法。该系统由100µm id×2 cm SCX捕捉柱和75µm id×12 cm C18 RP分析柱组成。在进样步骤中，流经SCX捕集柱的流量被引导为废物，以便在高流速下装载大量样品。然后将结合在SCX-TRAP柱上的多肽用高盐缓冲液洗脱到RP分析柱上，然后以纳升的流速进行RP色层分离。实验结果表明，SCX捕集柱的分离性能优于C18捕集柱。对牛血清白蛋白和酵母细胞裂解物的胰酶分析表明，该方法具有很高的蛋白质组学覆盖率。此外，该系统还被应用于人肝癌细胞系SMMC-7721胰酶消化液的二维分离，用于大规模蛋白质组分析。该系统是全自动化的，只需对现有的MU LC-MS/MS系统进行最小程度的改动。该系统为常规蛋白质组分析提供了一个很有前景的平台。
An approach was developed to automate sample introduction for nanoflow LC-MS/MS (mu LC-MS/ MS) analysis using a strong cation exchange (SCX) trap column. The system consisted of a 100 mu m id x 2 cm SCX trap column and a 75 mu m id x 12 cm C18 RP analytical column. During the sample loading step, the flow passing through the SCX trap column was directed to waste for loading a large volume of sample at high flow rate. Then the peptides bound on the SCX trap column were eluted onto the RP analytical column by a high salt buffer followed by RP chromatographic separation of the peptides at nanoliter flow rate. It was observed that higher performance of separation could be achieved with the system using SCX trap column than with the system using C18 trap column. The high proteomic coverage using this approach was demonstrated in the analysis of tryptic digest of BSA and yeast cell lysate. in addition, this system was also applied to two-dimensional separation of tryptic digest of human hepatocellular carcinoma cell line SMMC-7721 for large scale proteome analysis. This system was fully automated and required minimum changes on current mu LC-MS/MS system. This system represented a promising platform for routine proteome analysis.